PROJECT SUMMARY ABSTRACT Rates of neonatal abstinence syndrome have reached a staggering 6.5 per 1,000 births nationwide and, according to the CDC, Vermont, at 48.6 per 1,000, has the highest rate of women with opioid use disorder at delivery in the US. This situation creates an urgent need to identify how in utero exposure to opioids and associated risk factors influence the developing brain. Rigorous scientific investigation of this topic requires addressing the lack of foundational knowledge of neurotypical development. In addition, because postnatal brain development is modified by environmental experiences, it will be important to tease apart effects of prental opioid exposure from lifestyle factors associated with opioid use. This proposal brings together a multidisciplinary team with the expertise, experience, and institutional support to address these challenges in Vermont, a rural state hit particularly hard by the opioid epidemic. Through linking with three other sites, Oregon Health and Sciences University, New York University, and University of Pittsburgh Medical Center, the impact of this project is significantly enhanced. The activities in thie Phase I application will provide critical information to support a national level effort for Phase II of the HEALthy Brain and Child Development Study. In Specific Aim 1, we develop, implement and evaluate innovative recruitment and retention strategies for high- risk populations. This includes measuring the feasibility, throughput, and demographic characteristics associated with different recruitment methods through a longitudinal survey of 150 pregnant women per site (n=600 across sites), half of whom use opioids during pregnancy. Specific Aim 2 addresses anticipated challenges of the planned Phase II study by implementing and evaluating a multi-site, standardized, research protocol including multimodal MRI of placenta, fetus, neonate and 24-month-old brain, biospecimen collection, and asessment of substance use, and other key domains. Feasibility, data quality, and efficacy of different assessment methods will be evaluated in a longitudinal sample of 20 mother-infant dyads (N=80 across sites, half of whom use opioids during pregnancy), and a cross-sectional sample of five 24-month-old children with a history of prenatal opioid exposure (N=20 across sites). Specific Aim 3 evaluates data acquisition, processing, and statistical considerations to maximize data quality, usability, and integration across sites. In this aim we examine the efficacy of real-time motion monitoring for improving MRI data quality at different developmental stages; test novel MRI acceleration protocols; and harmonize data and processing pipelines for fetal, neonatal and child MRI into an open-source framework based on the universal Brain Imaging Data Structure (BIDS) and BIDS application routines. This project draws upon the investigators' foundational experience recruiting and retaining substance using pregnant women (MOTHER study; ECHO), working with our Vermont legal and ethical partners (i.e. IRB, Child Protective Services, etc.), and in collaborating in large muti-site neurodevelopmental studies (ABCD).